Efﬁcacy of In-Feed Tylosin Phosphate for the Treatment
of Necrotic Enteritis in Broiler Chickens
J. Brennan,* G. Moore,†,1
S. E. Poe,† A. Zimmermann,† G. Vessie,‡ D. A. Barnum,§ and J. Wilson||
*Shur-Gain Agresearch, R.R. 3, Burford, Ontario, Canada, N0E IA0; †Elanco Animal Health, Division of Eli Lilly and Co.,
Greenﬁeld, Indiana 46140; ‡Elanco, Division Eli Lilly Canada Inc., Research Park Centre, 150 Research Lane, Suite 120,
Guelph, Ontario, Canada N1G 4T2; and Departments of §Pathobiology, and ||Population Medicine,
Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1
ABSTRACT The efﬁcacy of tylosin phosphate for the
treatment of necrotic enteritis (NE) was investigated in a
ﬂoor pen study of 2,000 broiler chickens. A model in
which Clostridium perfringens was administered in the
feed on Days 14 to 16 was used to initiate an outbreak
of NE. Treatments, allocated at the pen level in a random-
ized complete block design, consisted of ﬁve levels of
tylosin phosphate (0, 50, 100, 200, or 300 ppm) adminis-
tered in the feed on Days 15 to 22, following the identiﬁ-
cation of an outbreak of NE on Day 15. Mortality due to
NE was signiﬁcantly reduced (P < 0.05) for medicated
birds at all dose levels of tylosin phosphate compared to
(Key words: tylosin phosphate, necrotic enteritis, broiler chicken, efﬁcacy)
2001 Poultry Science 80:1451–1454
Necrotic enteritis (NE), caused by the Gram-positive
bacillus Clostridium perfringens is an important disease of
broiler chickens, causing signiﬁcant morbidity and mor-
tality worldwide. The disease frequently occurs as out-
breaks characterized by depression, anorexia, and sudden
death. Control of this condition is dependent upon a vari-
ety of elements including reducing exposure to potential
dietary risk factors, reduction of concurrent enteric infec-
tions, particularly coccidiosis, and the use of feed addi-
tives with activity against C. perfringens (Ficken and
Although studies have demonstrated the efﬁcacy of
certain drugs in the prevention of NE (Long and Truscott,
1976; Prescott et al., 1978; George et al., 1982), few have
been demonstrated in feed to be effective in the treatment
of NE once clinical signs and intestinal lesions have be-
come apparent in affected birds (Hamdy et al., 1983a,b).
The macrolide antibiotic tylosin has been shown to
have in vitro activity against C. perfringens (Stutz and
2001 Poultry Science Association, Inc.
Received for publication December 11, 2000.
Accepted for publication June 15, 2001.
To whom correspondence should be addressed: moore_gregory_m@
unmedicated birds. Mean NE lesion scores on Day 17
were signiﬁcantly reduced (P < 0.05) by all levels of tylosin
treatment compared to those of unmedicated birds, de-
creasing linearly from 2.66 at 0 ppm to 0.38 at 100 ppm
and 0 at higher doses. Tylosin at all levels provided im-
provement in Day 29 body weight, average daily gain,
feed to gain ratio, and average daily feed intake compared
to unmedicated birds. The results of this study provide
evidence that tylosin phosphate, when administered in
feed, is effective in the treatment of clinical outbreaks of
NE in broiler chickens and suggest that the optimal dose
for this purpose is 100 ppm.
Lawton, 1984; Kondo, 1988; Watkins et al., 1997) and
to reduce the incidence of mortality associated with C.
perfringens enterotoxaemia in an intraduodenal C. per-
fringens inoculation model (Vissiennon et al., 2000). The
objective of this study was to evaluate the efﬁcacy of
tylosin phosphate administered in the feed for the treat-
ment of NE in broiler chickens during an induced clinical
outbreak mimicking typical conditions of modern poul-
MATERIALS AND METHODS
A total of 2,000 1-d-old Cobb × Cobb broiler chicks
obtained from a commercial hatchery were used in this
study. Birds were vaccinated for Marek’s disease at the
hatchery but received no other medications throughout
the study, other than tylosin phosphate. Replacements
were not made for early mortality. The research facility
was thoroughly cleaned and disinfected prior to bird
placement. Treatment groups were randomly assigned to
40 pens on concrete ﬂoors with new wood shavings for
bedding. The area of each pen was 4.2 m2
, and each
contained 25 male and 25 female birds at trial commence-
Abbreviation Key: NE = necrotic enteritis.
BRENNAN ET AL.1452
ment. Pens were separated by solid plastic barriers ex-
tending 30 cm up from ﬂoor level followed by 90 cm of
welded wire. Lighting program, heating, ventilation, and
other management procedures were typical of modern
intensive broiler farms in Ontario, Canada. Water was
provided ad libitum by four nipple-type drinkers per pen.
Dry feed was provided ad libitum by one tube-type feeder
per pen, except for a 12-h period of feed withdrawal
immediately prior to C. perfringens inoculum administra-
tion and during the challenge period itself, when inocu-
lum-feed mixture was provided in trough-type feeders.
All experimental procedures were conducted in accor-
dance with the guidelines of the Canadian Council on
Animal Care (1993).
A C. perfringens challenge model, based on that devel-
oped originally by Prescott et al. (1978), was used to initi-
ate an outbreak of NE among the experimental animals.
The model, with minor modiﬁcations, has been described
in a number of subsequent publications (Brennan et al.,
1996; Skinner and Brennan, 1999). Inoculum was adminis-
tered via the feed commencing Day 14 and ending Day
16. Birds were fed a commerical wheat-based broiler
starter diet from Days 0 to 21, except for Days 8 to 13
when a high protein starter containing 50% ﬁshmeal (a
recognized risk factor for naturally occurring outbreaks
of NE) (Truscott and Al-Sheikhly, 1977) was fed. A com-
mercial broiler grower diet was fed following the termina-
tion of the treatment period on Day 22 until trial termina-
tion on Day 29.
The challenge inoculum contained between 2 × 108
3 × 108
cfu C. perfringens/mL at administration. The inocu-
lum was prepared from a culture of C. perfringens isolated
from a bird displaying severe lesions of NE.
Birds that were moribund and unable to reach food or
water were culled and euthanized by cervical dislocation.
All dead or culled birds were necropsied to determine
the cause of morbidity or death. A gross pathologic diag-
nosis of NE was based on the presence of intestinal lesions
typical of naturally occurring and experimentally pro-
duced NE. These lesions consisted of focal to conﬂuent
areas of bland mucosal necrosis of the small intestine,
often forming a pseudomembrane with no gross evidence
of inﬂammatory reaction or hemorrhage. All dead or
culled birds were also scored for intestinal lesions of NE
as follows: 0 = no gross lesions; 1 = thin walled or friable;
2 = focal necrosis or ulceration; 3 = large patches of necro-
sis; 4 = severe extensive necrosis (Prescott et al., 1978). In
addition, on Day 15 one male bird per pen was euthanized
with inhaled carbon dioxide gas, subjected to necropsy,
and scored for gross lesions of NE and coccidiosis, the
latter according to criteria established for ﬂoor-pen stud-
ies in chickens (Johnson and Reid, 1970).
Criteria for declaring an NE outbreak were established
prior to commencement of the study and consisted of the
following: 1) observation of clinical signs in the ﬂock
consistent with naturally occurring and experimentally
produced outbreaks of NE (consisting of some combina-
tion of depression, huddling, reduced feed intake, and
changes in fecal consistency), and 2) at least 30% of birds
randomly selected on Day 15 for euthanizing having gross
intestinal lesions typical of NE as deﬁned above, or 3) at
least 30% of randomly selected and euthanized birds and
spontaneous mortalities postinoculation (and prior to ini-
tiation of treatments) combined having gross intestinal
lesions of NE.
On Day 15 an outbreak of NE was declared based on
the following criteria: 1) the ﬂock had clinical signs (as
described above) consistent with an outbreak of NE, 2)
36/40 (90%) randomly selected and euthazied birds had
gross intestinal lesions consistent with NE and no gross
evidence of coccidiosis, and 3) 5/6 (83%) birds found
dead on Day 15 had gross intestinal lesions consistent
Following identiﬁcation of the outbreak on Day 15,
treatments consisting of ﬁve levels of tylosin phosphate
(0, 50, 100, 200, or 300 ppm), administered in the feed for
seven consecutive days (Days 15 to 22), were randomly
allocated at the pen level in a randomized complete block
design. Each of the eight blocks consisted of ﬁve adjacent
pens within the trial facility. The investigators and techni-
cal staff were not informed of the treatment assignment
until the termination of the study.
Four birds were randomly selected from each pen (two
of each sex, total 160 birds) on each of d 17 and 22,
euthanized, and scored for gross lesions of NE and coccid-
iosis, as described above. Birds that died or were euthan-
ized for humane purposes postchallenge (Day 14) were
submitted for detection of C. perfringens. Gram-stained
smears of intestine were examined for the presence of
typical organisms; samples of the same tissues were cul-
tured anaerobically on blood agar at 37 C and examined
after 20 h for the presence of colonies exhibiting typical
growth characteristics (smooth, entire, and with a double
zone of hemolysis) (Ficken and Wages, 1997). Birds that
died or were euthanized for humane purposes after Day
14 were classiﬁed as positive for NE if they had gross
pathologic and microbiologic ﬁndings consistent with
Body weights were measured on a pen basis on Days
0 and 29. Feed consumption was measured on a pen basis
twice daily during the 3-d challenge and over the entire
study. One sample of each feed was analyzed for mois-
ture, crude protein, ash, fat, calcium, and phosphorus. A
TABLE 1. Mortality and lesion scores in a study of the efﬁcacy of
tylosin phosphate in the treatment of necrotic
enteritis (NE) in broiler chickens
Tylosin phosphate NE mortality, Mean NE lesion Mean NE lesion
(ppm) Days 15–29 (%) score, Days 17 score, Days 22
Means within a given column with no common superscript are sig-
niﬁcantly different (P < 0.05).
Day 22 mean NE lesions scores at 100 and 200 ppm are not signiﬁcantly
different from 0 ppm (P = 0.08).
EFFICACY OF TYLOSIN FOR TREATMENT OF NECROTIC ENTERITIS 1453
TABLE 2. Growth performance measures in a study of the efﬁcacy of tylosin phosphate
in the treatment of necrotic enteritis (NE) in broiler chickens
Tylosin phosphate Body weight, Average daily gain1
(ppm) Day 29 (kg) (kg/bird/day) Feed:gain1
Means within a given column with no common superscript are signiﬁcantly different (P < 0.05).
Adjusted for mortalities and removals due to morbidity.
sample of each feed was also analyzed for tylosin content
using HPLC. Permitted analytical variation of drug con-
tent for all medicated feeds was ± 25% of the calculated
Statistical analyses were conducted using the SAS Sys-
tem (SAS Institute, 1997). Necrotic enteritis pen mortality
data were analyzed by a series of pairwise treatment
comparisons using an exact P-value for the median test
(SAS Proc NPAR1WAY). For the purposes of this analysis,
spontaneous mortalities and moribund birds that were
euthanized for humane purposes and that had a ﬁnal
diagnosis of NE were treated identically. The Mann-Whit-
ney-Wilcoxon test (exact P-value from SAS Proc StatXact)
was used to conduct pairwise comparisons by treatment
of mean pen lesion scores. Analysis of variance was used
to test the effect of treatment on mean ﬁnal body weight,
average daily gain, ratio of feed to gain, and average daily
feed intake. The pen was considered the experimental
unit for all analyses.
All feeds were within tolerance for analyzed drug and
nutrient content. The addition of tylosin phosphate to the
feed signiﬁcantly reduced mortality (P < 0.05) due to NE
at all dose levels when compared to unmedicated birds
Mean NE lesion scores on Day 17 were signiﬁcantly
reduced (P < 0.05) by all levels of tylosin compared to
unmedicated birds, decreasing linearly from 2.66 at 0 ppm
to 0.38 at 100 ppm and 0 at higher doses (Table 1).
On Day 22, mean NE lesion scores were essentially
equal to or lower than those observed at corresponding
dose levels on Day 17. The mean NE lesion score observed
on Day 22 was signiﬁcantly lower (P < 0.05) for birds
administered tylosin at 300 ppm compared to unmedi-
cated birds (Table 1). Birds administered tylosin at 100
and 200 ppm had mean NE scores that were lower than
unmedicated birds but were not signiﬁcantly different (P
= 0.08). Coccidiosis lesion scores were 0 on Days 17 and
22 for all euthanized birds.
Tylosin at all levels provided improvement in Day 29
body weight, average daily gain, feed to gain ratio, and
average daily feed intake compared to unmedicated birds
The results of this study demonstrate that tylosin phos-
phate is effective in reducing morbidity, mortality, and
suppression of growth and feed efﬁciency associated with
NE in broiler chickens, when administered in the feed
after onset of an outbreak.
These ﬁndings are consistent with previous studies of
the activity of tylosin against C. perfringens in vitro (Stutz
and Lawton, 1984; Kondo, 1988; Watkins et al., 1997) and
support the ﬁnding of Vissiennon et al. (2000) that tylosin
is effective in reducing mortality associated with this
Signiﬁcantly reduced mean lesion scores among medi-
cated birds on Days 17 and 22 indicate that, in addition to
reducing mortality, administration of tylosin signiﬁcantly
reduced the clinical impact of challenge among surviving
birds. This result suggests that the positive effect of medi-
cation on growth parameters (Table 2) was related to
control of NE and not simply a result of a growth pro-
All dose levels of tylosin signiﬁcantly reduced mortal-
ity and mean lesion scores on Day 17 and improved
growth parameters. For Day 17, birds receiving 100 ppm
had signiﬁcantly reduced mean lesion scores compared
to those receiving 50 ppm. Although on Day 17, the birds
receiving 200 ppm dose had signiﬁcantly lower mean
lesion scores compared to those receiving 100 ppm, the
magnitude of the effect is not considered by the authors
to be of clinical relevance.
The results of this study thus provide evidence that
tylosin phosphate, when administered in feed, is effective
in the treatment of clinical outbreaks of NE in broiler
chickens and suggest that the optimal dose for this pur-
pose is 100 ppm.
Brennan, J. J., J. Radu, D. A. Barnum, D. Carrier, J. Kelly, and
B. Kilmer, 1996. Efﬁcacy of zinc bacitracin and bacitracin
methylene disalicyclate for prevention of necrotic enteritis
in broiler chickens. Poultry Sci. 75(Suppl. 1):283. (Abstr.).
Canadian Council on Animal Care, 1993. Guide to the Care and
Use of Experimental Animals. Vol. 1. 2nd ed. E. D. Olfert,
B. M. Cross, and A. A. McWilliam, ed. CCAC Ottawa,
BRENNAN ET AL.1454
Ficken, M. D., and D. P. Wages, 1997. Necrotic enteritis. Pages
261–264 in: Diseases of Poultry. 10th ed. B. W. Calnek, ed.
Iowa State University Press, Ames, IA.
George, B. A., C. L. Quarles, and D. J. Fagerberg, 1982. Virgin-
iamycin effects on controlling necrotic enteritis infection in
chickens. Poultry Sci. 61:447–450.
Hamdy, A. H., R. W. Thomas, D. D. Kratzer, and R. B. Davis,
1983a. Lincomycin dose response for treatment of necrotic
enteritis in broilers. Poultry Sci. 62:585–588.
Hamdy, A. H., R. W. Thomas, R. J. Yancey, and R. B. Davis,
1983b. Therapeutic effect of optimal lincomycin concentra-
tion in drinking water on necrotic enteritis in broilers. Poultry
Johnson, J. K., and W. M. Reid., 1970. Anticoccidial drugs: Lesion
scoring techniques in battery and ﬂoor-pen experiments with
chickens. Exp. Parasitol. 28:30–36.
Kondo, F., 1988. In vitro lecithinase activity and sensitivity to
22 antimicrobial agents on Clostridium perfringens isolated
from necrotic enteritis of broiler chickens. Res. Vet. Sci.
Long, J. R., and R. B. Truscott, 1976. Necrotic enteritis in broiler
chickens. III. Reproduction of the disease. Can. J. Comp. Med.
Prescott, J. F., R. Sivendra, and D. A. Barnum, 1978. The use of
bacitracin in the prevention and treatment of experimentally-
induced necrotic enteritis in the chicken. Can. Vet. J.
SAS Institute Inc., 1997. SAS/STAT Software: Changes and
Enhancements Through Release 6.12, SAS Institute Inc.,
Skinner, J,. and J. Brennan, 1999. Efﬁcacy of bacitracin methylene
disalicyclate fed in combination with narasin for prevention
of necrotic enteritis. Poultry Sci. 78(Suppl. 1):129. (Abstr.).
Stutz, M. W., and G. C. Lawton, 1984. Effects on diet and antimi-
crobials on growth, feed efﬁciency, intestinal Clostridium per-
fringens, and ileal weight of broiler chicks. Poultry Sci.
Truscott, R. B., and F. Al-Sheikhly, 1977. Reproduction and
treatment of necrotic enteritis in broilers. Am. J. Vet. Res.
Vissiennon, Th., H. Kro¨ger, T. Ko¨hler, and R. Kliche, 2000. Effect
of avilamycin, tylosin and ionophore anticoccidials on Clos-
tridium perfringens enterotoxaemia in chickens. Berl. Munch.
Tiera¨rztl. Wschr. 113:9–13.
Watkins, K. L., T. R. Shryock, R. N. Dearth, and Y. M. Saif, 1997.
In vitro antimicrobial susceptibility of Clostridium perfringens
from commercial turkey and broiler chicken origin. Vet. Mi-